Intergenic regions as insertion sites in the genome of modified vaccinia virus ankara (MVA)

ABSTRACT

The present invention relates to novel insertion sites useful for the integration of exogenous sequences into the Modified Vaccinia Ankara (MVA) virus genome. The present invention further provides plasmid vectors to insert exogenous DNA into the genome of MVA. Furthermore, the present invention provides recombinant MVA comprising an exogenous DNA sequence inserted into the new insertion site as medicine or vaccine.

This application is a continuation of U.S. application Ser. No.12/468,120 (now U.S. Pat. No. 7,964,374), filed on May 19, 2009, whichis a divisional of U.S. application Ser. No. 10/514,761 (now U.S. Pat.No. 7,550,147), filed on Nov. 16, 2004, which was the National Stage ofInternational Application PCT/EP03/05045, filed May 14, 2003, whichclaims the benefit of PA 2002 00752, filed on May 16, 2002, in Denmarkand PA 2002 00753, filed on May 16, 2002, in Denmark, all of which areincorporated herein by reference.

The present invention relates to novel insertion sites useful for theintegration of exogenous DNA sequences into the MVA genome.

BACKGROUND OF THE INVENTION

Modified Vaccinia Virus Ankara (MVA) is a member of the Orthopoxvirusfamily and has been generated by about 570 serial passages on chickenembryo fibroblasts of the Ankara strain of Vaccinia virus (CVA) (forreview see Mayr, A., et al. [1975], Infection 3, 6-14). As a consequenceof these passages the resulting MVA virus contains 31 kilobases lessgenomic information compared to CVA and is highly host cell restricted(Meyer, H. et al., J. Gen. Virol. 72, 1031-1038 [1991]). MVA ischaracterized by its extreme attenuation, namely by a diminishedvirulence or infectiosity but still an excellent immunogenicity. Whentested in a variety of animal models, MVA was proven to be avirulenteven in immuno-suppressed individuals. More importantly, the excellentproperties of the MVA strain have been demonstrated in extensiveclinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167,375-390 [1987]). During these studies in over 120,000 humans, includinghigh risk patients, no side effects were seen (Stickl et al., Dtsch.med. Wschr. 99, 2386-2392 [1974]).

It has been further found that MVA is blocked in the late stage of thevirus replication cycle in mammalian cells (Sutter, G. and Moss, B.[1992] Proc. Natl. Acad. Sci. USA 89, 10847-10851). Accordingly, MVAfully replicates its DNA, synthesizes early, intermediate and late geneproducts, but is not capable to assemble mature infectious virions,which could be released from an infected cell. For this reason, namelyto be replication restricted, MVA was proposed to serve as a geneexpression vector.

More recently, MVA was used to generate recombinant vaccines, expressingantigenic sequences inserted either at the site of the tymidine-kinase(tk) gene (U.S. Pat. No. 5,185,146) or at the site of a naturallyoccurring deletion within the MVA genome (PCT/EP96/02926).

Although the tk insertion locus is widely used for the generation ofrecombinant poxviruses, particularly for the generation of recombinantVaccinia viruses (Mackett, et al. [1982] P.N.A.S. USA 79, 7415-7419)this technology was not applicable for MVA. It was shown by Scheiflingeret al., that MVA is much more sensitive to modifications of the genomecompared to other poxviruses, which can be used for the generation ofrecombinant poxviruses. Scheiflinger et al. showed in particular thatone of the most commonly used site for the integration of heterologousDNA into poxyiral genomes, namely the thymdine kinase (tk) gene locus,cannot be used to generate recombinant MVA. Any resulting tk(−)recombinant MVA proved to be highly unstable and upon purificationimmediately deleted the inserted DNA together with parts of the genomicDNA of MVA (Scheiflinger et al. [1996], Arch Virol 141: pp 663-669).

Instability and, thus, high probability of genomic recombination is aknown problem within pox virology. Actually, MVA was established duringlong-term passages exploiting the fact that the viral genome of CVA isunstable when propagated in vitro in tissue cultured cells. Severalthousands of nucleotides (31 kb) had been deleted from the MVA genome,which therefore is characterized by 6 major and numerous small deletionsin comparison to the original CVA genome.

The genomic organization of the MVA genome has been described recently(Antoine et al. [1998], Virology 244, 365-396). The 178 kb genome of MVAis densely packed and comprises 193 individual open reading frames(ORFs), which code for proteins of at least 63′ amino acids in length.In comparison with the highly infectious Variola virus and also theprototype of Vaccinia virus, namely the strain Copenhagen, the majorityof ORFs of MVA are fragmented or truncated (Antoine et al. [1998],Virology 244, 365-396). However, with very few exceptions all ORFs,including the fragmented and truncated ORFs, get transcribed andtranslated into proteins. In the following, the nomenclature of Antoineet al. is used and—where appropriate—the nomenclature based on Hind IIIrestriction enzyme digest is also indicated.

So far, only the insertion of exogenous DNA into the naturally occurringdeletion sites of the MVA genome led to stable recombinant MVAs(PCT/EP96/02926). Unfortunately, there is only a restricted number ofnaturally occurring deletion sites in the MVA genome. Additionally itwas shown that other insertion sites, such as, e.g., the tk gene locus,are hardly useful for the generation of recombinant MVA (Scheiflinger etal. [1996], Arch Virol 141: pp 663-669).

OBJECT OF THE INVENTION

It is an object of the present invention to identify further insertionsites of the MVA genome and to provide insertion vectors, which directthe insertion of exogenous DNA sequences into said newly identifiedinsertion sites of the MVA genome.

It is a further object of the present invention to provide a recombinantMVA, which comprises exogenous DNA sequences stably integrated into newinsertion sites of the MVA genome.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention identified new sites for theinsertion of exogenous DNA sequences into the genome of ModifiedVaccinia Ankara (MVA) virus. The new insertion sites are located in theintergenic regions (IGRs) of the viral genome, wherein said IGRs are, inturn, located between or are flanked by two adjacent open reading frames(ORFs) of the MVA genome.

Accordingly, the present invention relates to a recombinant MVAcomprising a heterologous DNA sequence inserted into an IGR of the viralgenome. According to the present invention, one or more exogenous DNAsequences may be inserted into one or more IGRs.

It was surprisingly found that exogenous DNA sequences remain indeedstable inserted into IGRs of the MVA genome: As already indicated above,the genome of MVA is to be considered as being quite unstable. It seemsthat genes or DNA sequences non-essential for propagation of the virusare deleted or fragmented. Although it was—also surprisingly—found thatstable recombinant MVAs are obtained when heterologous DNA sequences areinserted into the naturally occurring deletion sites of the MVA genome(PCT/EP96/02926) it was—on the other hand—found that host range genesas, e.g., the tk-locus widely used for the generation of otherrecombinant poxviruses are no suitable insertion sites in MVA. The factthat Vero-MVA has one extra genomic deletion (PCT/EP01/02703) alsosuggests that the genome is dynamic in the sense it readily deletesgenes that are not required for propagation. Therefore, it could beconcluded that inserting heterologous DNA sequences non-essential forviral propagation into spaces between ORFs would be expected to bedeleted by the virus as well.

While the nucleotide sequence of an ORF encodes an amino acid sequenceforming a peptide, polypeptide or protein, the IGRs between two ORFshave no coding capacity, but may comprise regulatory elements, bindingsites, promoter and/or enhancer sequences essential for or involved inthe transcriptional control of the viral gene expression. Thus, the IGRmay be involved in the regulatory control of the viral life cycle.However, the inventors of the present invention have also shown that thenew insertion sites have the unexpected advantage that exogenous DNAsequences can be stably inserted into the MVA genome without influencingor changing the typical characteristics and gene expression of MVA. Thenew insertion sites are especially useful, since no ORF or codingsequence of MVA is altered.

Moreover, it was surprisingly found that the expression level of aforeign gene inserted into an IGR is higher than the expression level ofa foreign gene inserted into a deletion site of the MVA genome (see alsoExample 1).

The nucleotide sequence of an ORF regularly starts with a start codonand ends with a stop codon. Depending on the orientation of the twoadjacent ORFs the IGR, the region in between these ORFs, is flankedeither by the two stop codons of the two adjacent ORFs, or, by the twostart codons of the two adjacent ORFs, or, by the stop codon of thefirst ORF and the start codon of the second ORF, or, by the start codonof the first ORF and the stop codon of the second ORF.

Accordingly, the insertion site for the exogenous DNA sequence into theIGR may be downstream or 3′ of the stop codon of a first ORF. In casethe adjacent ORF, also termed second ORF, has the same orientation asthe first ORF, this insertion site downstream of the stop codon of thefirst ORF lies upstream or 5′ of the start codon of the second ORF.

In case the second ORF has an opposite orientation relative to the firstORF, which means the orientation of the two adjacent ORFs points to eachother, then the insertion site lies downstream of the stop codons ofboth ORFs.

As a third alternative, in case the two adjacent ORFs read in oppositedirection, but the orientation of the two adjacent ORFs points away fromeach other, which is synonymous with a positioning that is characterizedin that the start codons of the two ORFs are adjacent to each other,then the exogenous DNA is inserted upstream relative to both startcodons.

ORFs in the MVA genome occur in two coding directions. Consequently, thePolymerase activity occurs from left to right, i.e., forward directionand, correspondingly, from right to left (reverse direction). It iscommon practice in poxvirology and it became a standard classificationfor Vaccinia viruses to identify ORFs by their orientation and theirposition on the different HindIII restriction digest fragments of thegenome. For the nomenclature, the different HindIII fragments are namedby descending capital letters corresponding with their descending size.The ORF are numbered from left to right on each HindIII fragment and theorientation of the ORF is indicated by a capital L (standing fortranscription from right to Left) or R (standing for transcription fromleft to Right). Additionally, there is a more recent publication of theMVA genome structure, which uses a different nomenclature, simplynumbering the ORF from the left to the right end of the genome andindicating their orientation with a capital L or R (Antoine et al.[1998], Virology 244, 365-396). As an example the I4L ORF, according tothe old nomenclature, corresponds to the 064L ORF according to Antoineet al. If not indicated differently, the present invention uses thenomenclature according to Antoine et al.

According to the present invention, heterologous DNA sequences can beinserted into one or more IGRs inbetween two adjacent ORFs selected fromthe group comprising:

001L-002L, 002L-003L, 005R-006R, 006L-007R, 007R-008L, 008L-009L,017L-018L, 018L-019L, 019L-020L, 020L-021L, 023L-024L, 024L-025L,025L-026L, 028R-029L, 030L-031L, 031L-032L, 032L-033L, 035L-036L,036L-037L, 037L-038L, 039L-040L, 043L-044L, 044L-045L, 046L-047R,049L-050L, 050L-051L, 051L-052R, 052R-053R, 053R-054R, 054R-055R,055R-056L, 061L-062L, 064L-065L, 065L-066L, 066L-067L, 077L-078R,078R-079R, 080R-081R, 081R-082L, 082L-083R, 085R-086R, 086R-087R,088R-089L, 089L-090R, 092R-093L, 094L-095R, 096R-097R, 097R-098R,101R-102R, 103R-104R, 105L-106R, 107R-108L, 108L-109L, 109L-110L,110L-111L, 113L-114L, 114L-115L, 115L-116R, 117L-118L, 118L-119R,122R-123L, 123L-124L, 124L-125L, 125L-126L, 133R-134R, 134R-135R,136L-137L, 137L-138L, 141L-142R, 143L-144R, 144R-145R, 145R-146R,146R-147R, 147R-148R, 148R-149L, 152R-153L, 153L-154R, 154R-155R,156R-157L, 157L-158R, 159R-160L, 160L-161R, 162R-163R, 163R-164R,164R-165R, 165R-166R, 166R-167R, 167R-168R, 170R-171R, 173R-174R,175R-176R, 176R-177R, 178R-179R, 179R-180R, 180R-181R, 183R-184R,184R-185L, 185L-186R, 186R-187R, 187R-188R, 188R-189R, 189R-190R,192R-193R.

According to the old nomenclature, ORF 006L corresponds to C10L, 019Lcorresponds to C6L, 020L to N1L, 021L to N2L, 023L to K2L, 028R to K7R,029L to F1L, 037L to F8L, 045L to F15L, 050L to E3L, 052R to E5R, 054Rto E7R, 055R to E8R, 056L to E9L, 062L to I1L, 064L to I4L, 065L to I5L,081R to L2R, 082L to L3L, 086R to J2R, 088R to J4R, 089L to J5L, 092R toH2R, 095R to H5R, 107R to D10R, 108L to D11L, 122R to A11R, 123L toA12L, 125L to A14L, 126L to A15L, 135R to A24R, 136L to A25L, 137L toA26L, 141L to A30L, 148R to A37R, 149L to A38L, 152R to A40R, 153L toA41L, 154R to A42R, 157L to A44L, 159R to A46R, 160L to A47L, 165R toA56R, 166R to A57R, 167R to B1R, 170R to B3R, 176R to B8R, 180R to B12R,184R to B16R, 185L to B17L, and 187R to B19R.

Preferably, the heterologous sequence is inserted into an IGR flanked bytwo adjacent ORFs selected from the group comprising 007R-008L,018L-019L, 044L-045L, 064L-065L, 136L-137L, 148R-149L.

Heterologous or exogenous DNA sequences are sequences which, in nature,are not normally found associated with the poxvirus as used according tothe present invention. According to a further embodiment of the presentinvention, the exogenous DNA sequence comprises at least one codingsequence. The coding sequence is operatively linked to a transcriptioncontrol element, preferably to a poxyiral transcription control element.Additionally, also combinations between poxyiral transcription controlelement and, e.g., internal ribosomal entry sites can be used.

According to a further embodiment, the exogenous DNA sequence can alsocomprise two or more coding sequences linked to one or severaltranscription control elements. Preferably, the coding sequence encodesone or more proteins, polypeptides, peptides, foreign antigens orantigenic epitopes, especially those of therapeutically interestinggenes.

Therapeutically interesting genes according to the present invention maybe genes derived from or homologous to genes of pathogenous orinfectious microorganisms which are disease causing. Accordingly, in thecontext of the present invention such therapeutically interesting genesare presented to the immune system of an organism in order to affect,preferably induce a specific immune response and, thereby, vaccinate orprophylactically protect the organism against an infection with themicroorganism. In further preferred embodiments of the present inventionthe therapeutically interesting genes are selected from genes ofinfectious viruses, e.g., —but not limited to—Dengue virus, Japaneseencephalitis virus, Hepatitis virus B or C, or immunodeficiency virusessuch as HIV.

Genes derived from Dengue virus are preferably NS1 and PrM genes,wherein said genes may be derived from one, two, three or from all ofthe 4 Dengue virus serotypes. The NS1 gene is preferably derived fromDengue virus serotype 2 and is preferably inserted into the IGR betweenthe ORFs 064L-065L (I4L-I5L). PrM genes, preferably derived from all ofthe 4 Dengue virus serotypes, are preferably inserted into the IGRsbetween the ORFs selected from 007R-008L, 044L-045L, 136L-137L,148R-149L. More preferably, the PrM gene derived from Dengue virusserotype 1 (prM 1) is inserted into the IGR of 148R-149L, PrM 2 into theIGR 007R-008L, PrM 3 into the IGR of the ORFs 044L-045L, and PrM 4 intothe IGR 136L-137L.

According to a further preferred embodiment of the present invention theheterologous DNA sequence is derived from HIV and encodes HIV env,wherein the HIV env gene is preferably inserted into the IGR between theORFs 007R-008L.

Furthermore, therapeutically interesting genes according to the presentinvention also comprise disease related genes, which have a therapeuticeffect on proliferative disorder, cancer or metabolic diseases. Forexample, a therapeutically interesting gene regarding cancer could be acancer antigen that has the capacity to induce a specific anti-cancerimmune reaction.

According to a further embodiment of the present invention, the codingsequence comprises at least one marker or selection gene.

Selection genes transduce a particular resistance to a cell, whereby acertain selection method becomes possible. The skilled practitioner isfamiliar with a variety of selection genes, which can be used in apoxyiral system. Among these are, e.g., Neomycin resistance gene (NPT)or Phosphoribosyl transferase gene (gpt).

Marker genes induce a colour reaction in transduced cells, which can beused to identify transduced cells. The skilled practitioner is familiarwith a variety of marker genes, which can be used in a poxyiral system.Among these are the gene encoding, e.g., β-Galactosidase (β-gal),β-Glucosidase (β-glu), Green Fluorescence protein (EGFP) or BlueFluorescence Protein.

According to still a further embodiment of the present invention theexogenous DNA sequence comprises a spacing sequence, which separatespoxyiral transcription control element and/or coding sequence in theexogenous DNA sequence from the stop codon and/or the start codon of theadjacent ORFs. This spacer sequence between the stop/start codon of theadjacent ORF and the inserted coding sequence in the exogenous DNA hasthe advantage to stabilize the inserted exogenous DNA and, thus, anyresulting recombinant virus. The size of the spacer sequence is variableas long as the sequence is without own coding or regulatory function.

According to a further embodiment, the spacer sequence separating thepoxyiral transcription control element and/or the coding sequence in theexogenous DNA sequence from the stop codon of the adjacent ORF is atleast one nucleotide long.

According to another embodiment of the present invention, the spacingsequence separating the poxyiral transcription control element and/orthe coding sequence in the exogenous DNA sequence from the start codonof the adjacent ORF is at least 30 nucleotides. Particularly, in caseswhere a typical Vaccinia virus promoter element is identified upstreamof a start codon the insertion of exogenous DNA may not separate thepromoter element from the start codon of the adjacent ORF. A typicalVaccinia promoter element can be identified by scanning for e.g. thesequence “TAAAT” for late promoters (Davison & Moss, J. Mol. Biol. 1989;210: 771-784) and an A/T rich domain for early promoters. A spacingsequence of about 30 nucleotides is the preferred distance to securethat a poxyiral promoter located upstream of the start codon of the ORFis not influenced. Additionally, according to a further preferredembodiment, the distance between the inserted exogenous DNA and thestart codon of the adjacent ORF is around 50 nucleotides and morepreferably around 100 nucleotides.

According to a further preferred embodiment of the present invention,the spacing sequence comprises an additional poxyiral transcriptioncontrol element which is capable to control the transcription of theadjacent ORF.

A typical MVA strain which can be used according to the presentinvention for generating a recombinant MVA is MVA-575 that has beendeposited at the European Collection of Animal Cell Cultures under thedeposition number ECACC V00120707.

Another preferred MVA strain is MVA-Vero or a derivative thereof.MVA-Vero strains have been deposited at the European Collection ofAnimal Cell Cultures under the deposition numbers ECACC V99101431 and01021411. The safety of the MVA-Vero is reflected by biological,chemical and physical characteristics as described in the InternationalPatent Application PCT/EP01/02703. In comparison to other MVA strains,the Vero-MVA includes one additional genomic deletion.

Still another, more preferred MVA strain is MVA-BN. MVA-BN has beendeposited at the European Collection of Animal Cell Cultures with thedeposition number ECACC V00083008. MVA-BN virus is an extremelyattenuated virus also derived from Modified Vaccinia Ankara virus (seealso PCT/EP01/13628).

The term “derivatives” of a virus according to the present inventionrefers to progeny viruses showing the same characteristic features asthe parent virus but showing differences in one or more parts of itsgenome. The term “derivative of MVA” describes a virus, which has thesame functional characteristics compared to MVA. For example, aderivative of MVA-BN has the characteristic features of MVA-BN. One ofthese characteristics of MVA-EN or derivatives thereof is itsattenuation and lack of replication in human HaCat cells.

The recombinant MVA according to the present invention is useful as amedicament or vaccine. It is, according to a further embodiment, usedfor the introduction of the exogenous coding sequence into a targetcell, said sequence being either homologous or heterologous to thegenome of the target cell.

The introduction of an exogenous coding sequence into a target cell maybe done in vitro to produce proteins, polypeptides, peptides, antigensor antigenic epitopes. This method comprises the infection of a hostcell with the recombinant MVA according to the invention, cultivation ofthe infected host cell under suitable conditions, and isolation and/orenrichment of the polypeptide, peptide, protein, antigen, epitope and/orvirus produced by said host cell.

Furthermore, the method for introduction of one or more homologous orone or more heterologous sequence into cells may be applied for in vitroand in vivo therapy. For in vitro therapy, isolated cells that have beenpreviously (ex vivo) infected with the recombinant MVA according to theinvention are administered to the living animal body for affecting,preferably inducing an immune response. For in vivo therapy, therecombinant poxvirus according to the invention is directly administeredto the living animal body for affecting, preferably inducing an immuneresponse. In this case, the cells surrounding the site of inoculation,but also cells where the virus is transported to via, e.g., the bloodstream, are directly infected in vivo by the recombinant MVA accordingto the invention. After infection, these cells synthesize the proteins,peptides or antigenic epitopes of the therapeutic genes, which areencoded by the exogenous coding sequences and, subsequently, presentthem or parts thereof on the cellular surface. Specialized cells of theimmune system recognize the presentation of such heterologous proteins,peptides or epitopes and launch a specific immune response.

Since the MVA is highly growth restricted and, thus, highly attenuated,it is useful for the treatment of a wide range of mammals includinghumans, including immune-compromised animals or humans. The presentinvention also provides pharmaceutical compositions and vaccines forinducing an immune response in a living animal body, including a human.

The pharmaceutical composition may generally include one or morepharmaceutical acceptable and/or approved carriers, additives,antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Suchauxiliary substances can be water, saline, glycerol, ethanol, wetting oremulsifying agents, pH buffering substances, or the like. Suitablecarriers are typically large, slowly metabolized molecules such asproteins, polysaccharides, polylactic acids, polyglycollic acids,polymeric amino acids, amino acid copolymers, lipid aggregates, or thelike.

For the preparation of vaccines, the recombinant poxvirus according tothe invention is converted into a physiologically acceptable form. Thiscan be done based on the experience in the preparation of poxvirusvaccines used for vaccination against smallpox (as described by Stickl,H. et al. [1974] Dtsch. med. Wschr. 99, 2386-2392). For example, thepurified virus is stored at −80° C. with a titre of 5×10E8 TCID₅₀/mlformulated in about 10 mM Tris, 140 mM NaCl pH 7.4. For the preparationof vaccine shots, e.g., 10E2-10E8 particles of the virus are lyophilizedin 100 ml of phosphate-buffered saline (PBS) in the presence of 2%peptone and 1% human albumin in an ampoule, preferably a glass ampoule.Alternatively, the vaccine shots can be produced by stepwisefreeze-drying of the virus in a formulation. This formulation cancontain additional additives such as mannitol, dextran, sugar, glycine,lactose or polyvinylpyrrolidone or other aids such as antioxidants orinert gas, stabilizers or recombinant proteins (e.g. human serumalbumin) suitable for in vivo administration. The glass ampoule is thensealed and can be stored between 4° C. and room temperature for severalmonths. However, as long as no need exists the ampoule is storedpreferably at temperatures below −20° C.

For vaccination or therapy the lyophilisate can be dissolved in 0.1 to0.5 ml of an aqueous solution, preferably physiological saline or Trisbuffer, and administered either systemically or locally, i.e.parenterally, subcutaneous, intramuscularly, by scarification or anyother path of administration know to the skilled practitioner. The modeof administration, the dose and the number of administrations can beoptimized by those skilled in the art in a known manner. However, mostcommonly a patient is vaccinated with a second shot about one month tosix weeks after the first vaccination shot.

The present invention further relates to plasmid vectors, which can beused to generate recombinant MVA according to the present invention, andalso relates to certain DNA sequences:

Regularly, the IGR located between or flanked by two adjacent ORFscomprises nucleotide sequences in which the exogenous DNA sequence ofinterest can be inserted. Accordingly, the plasmid vector according tothe present invention comprises a DNA sequence derived from orhomologous to the genome of MVA, wherein said DNA sequence comprises acomplete or partial fragment of an IGR sequence located between orflanked by two adjacent ORFs of the viral genome. Preferably, theplasmid vector comprises inserted into said IGR-derived sequence atleast one cloning site for the insertion of an exogenous DNA sequence ofinterest and, preferably, for the insertion of a poxyiral transcriptioncontrol element operatively linked to said heterologous DNA sequence.Optionally, the plasmid vector comprises a reporter- and/or selectiongene cassette. The plasmid vector preferably also comprises sequences ofthe two adjacent ORFs flanking said complete or partial fragment of theIGR sequence.

Some IGRs have been identified which do not include nucleotidesequences. In these cases, the plasmid vector comprises DNA sequences ofthe IGR flanking sequences, i.e., DNA sequences of the two adjacentORFs. Preferably, the cloning site for the insertion of the heterologousDNA sequence is inserted into the IGR. The DNA of the IGR flankingsequences is used to direct the insertion of exogenous DNA sequencesinto the corresponding IGR in the MVA genome. Such a plasmid vector mayadditionally include a complete or partial fragment of an IGR sequencewhich comprises the cloning site for the insertion of the heterologousDNA sequence and, optionally, of the reporter- and/or selection genecassette.

IGR-DNA sequences as well as IGR flanking sequences of the two adjacentORFs are preferably selected from IGRs and ORFs, respectively, selectedfrom the group comprising

001L-002L, 002L-003L, 005R-006R, 006L-007R, 007R-008L, 008L-009L,017L-018L, 018L-019L, 019L-020L, 020L-021L, 023L-024L, 024L-025L,025L-026L, 028R-029L, 030L-031L, 031L-032L, 032L-033L, 035L-036L,036L-037L, 037L-038L, 039L-040L, 043L-044L, 044L-045L, 046L-047R,049L-050L, 050L-051L, 051L-052R, 052R-053R, 053R-054R, 054R-055R,055R-056L, 061L-062L, 064L-065L, 065L-066L, 066L-067L, 077L-078R,078R-079R, 080R-081R, 081R-082L, 082L-083R, 085R-086R, 086R-087R,088R-089L, 089L-090R, 092R-093L, 094L-095R, 096R-097R, 097R-098R,1018-102R, 103R-104R, 105L-106R, 107R-108L, 108L-109L, 109L-110L,110L-111L, 113L-114L, 114L-115L, 115L-116R, 117L-118L, 118L-119R,122R-123L, 123L-124L, 124L-125L, 125L-126L, 133R-134R, 134R-135R,136L-137L, 137L-138L, 141L-142R, 143L-144R, 144R-145R, 145R-146R,146R-147R, 147R-148R, 148R-149L, 152R-153L, 153L-154R, 154R-155R,156R-157L, 157L-158R, 159R-160L, 160L-161R, 162R-163R, 163R-164R,164R-165R, 165R-166R, 166R-167R, 167R-168R, 170R-171R, 173R-174R,175R-176R, 176R-177R, 178R-179R, 179R-180R, 180R-181R, 183R-184R,184R-185L, 185L-186R, 186R-187R, 187R-188R, 188R-189R, 189R-190R,192R-193R.

The sequences are, more preferably, selected from IGRs and ORFs,respectively, selected from the group comprising 007R-008L, 018L-019L,044L-045L, 064L-065L, 136L-137L, 148L-149L. IGR derived sequences are,preferably, selected from the group comprising the nucleotide sequences

-   -   no. 527-608 of SeqID No. 32;    -   no. 299-883 of SeqID No. 33;    -   no. 339-852 of SeqID No. 34;    -   no. 376-647 of SeqID No. 35;    -   no. 597-855 of SeqID No. 36;    -   no. 400-607 of SeqID No. 37.

IGR flanking sequences of the two adjacent ORFs are, preferably,selected from the group comprising the nucleotide sequences:

-   -   no. 1-525 and 609-1190 of SeqID No. 32;    -   no. 101-298 and 884-1198 of SeqID No. 33;    -   no. 1-338 and 853-1200 of SeqID No. 34;    -   no. 1-375 and 648-1200 of SeqID No. 35;    -   no. 1-596 and 856-1200 of SeqID No. 36;    -   no. 1-399 and 608-1081 of SeqID No. 37.

The DNA sequences are preferably derived from or homologous to thegenome of the MVA deposited at ECACC under deposition number V00083008.

To generate a plasmid vector according to the present invention thesequences are isolated and cloned into a standard cloning vector, suchas pBluescript (Stratagene), wherein they flank the exogenous DNA to beinserted into the MVA genome. Optionally, such a plasmid vectorcomprises a selection- or reporter gene cassette, which can be deletedfrom the final recombinant virus, due to a repetitive sequence includedinto said cassette.

Methods to introduce exogenous DNA sequences by a plasmid vector into anMVA genome and methods to obtain recombinant MVA are well known to theperson skilled in the art and, additionally, can be deduced from thefollowing references:

-   -   Molecular Cloning, A laboratory Manual. Second Edition. By J.        Sambrook, E. F. Fritsch and T. Maniatis. Cold Spring Harbor        Laboratory Press. 1989: describes techniques and know how for        standard molecular biology techniques such cloning of DNA, RNA        isolation, western blot analysis, RT-PCR and PCR amplification        techniques;    -   Virology Methods Manual. Edited by Brian W J Mahy and Hillar O        Kangro. Academic Press. 1996: describes techniques for the        handling and manipulation of viruses;    -   Molecular Virology: A Practical Approach. Edited by A J Davison        and R M Elliott. The Practical Approach Series. IRL Press at        Oxford University Press. Oxford 1993. Chapter 9: Expression of        genes by Vaccinia virus vectors;    -   Current Protocols in Molecular Biology. Publisher: John Wiley        and Son Inc. 1998. Chapter 16, section IV: Expression of        proteins in mammalian cells using Vaccinia viral vector:        describes techniques and know-how for the handling, manipulation        and genetic engineering of MVA.

The MVA according to the present invention, preferably the MVA depositedat ECACC under deposition number V00083008, may be produced bytransfecting a cell with a plasmid vector according to the presentinvention, infecting the transfected cell with an MVA and, subsequently,identifying, isolating and, optionally, purifying the MVA according tothe invention.

The DNA sequences according to the invention can be used to identify orisolate the MVA or its derivatives according to the invention and cellsor individuals infected with an MVA according to the present invention.The DNA sequences are, e.g., used to generate PCR-primers, hybridizationprobes or are used in array technologies.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: Restriction map of the vector constructs pBNX39 (FIG. 1 a),pBNX70 (FIG. 1 b) and pBN84 (FIG. 1 c), comprising about 600 bp of MVAsequences flanking the insertion site after the I4L ORF. The plasmidsadditionally comprise exogenous DNA (Ecogpt and hBFP, respectively)under the transcriptional control of a poxvirus promoter P) between theflanking sequences: Flank 1 (F1 I4L-I5L) and Flank 2 (F2 I4L-I5L). F1rptstands for a repetitive sequence of Flank 1 to allow deletion of thereporter cassette from a resulting recombinant virus. pBN84 (FIG. 1 c)additionally codes for the Denguevirus NS1 protein (NS1 DEN). Furtherabbreviations: AmpR=Ampicilin resistance gene; bps=base pairs.

FIG. 2: Restriction map of the vector constructs pBNX51 (FIG. 2 a),pBNX67 (FIG. 2 b) and pBN27 (FIG. 2 c), comprising about 600 bp of MVAsequences flanking the insertion site after the ORF 137L (Flank 1:F1136-137 corresponds to position 129340-129930 of the MVA genome; Flank2: F2136-137 corresponds to position 129931-130540 of the MVA genome).Additionally the vector pBNX67 (FIG. 2 b) comprises exogenous DNA (NPTII gene=neomycin resistance) under the transcriptional control of apoxvirus promoter P) between the flanking sequences. F2rpt stands for arepetitive sequence of Flank 2 to allow deletion of the reportercassette from a resulting recombinant virus. pBN27 (FIG. 2 c)additionally codes for the Denguevirus PrM4 under control of a poxviruspromoter. Further abbreviations: AmpR=Ampicilin resistance gene;bps=base pairs; IRES=internal ribosomal entry site; EGFP=gene for theenhanced green fluorescent protein.

FIG. 3: Restriction map of the vector constructs pBNX79 (FIG. 3 a),pBNX86 (FIG. 3 b), pBNX88, (FIG. 3 c), pBN34 (FIG. 3 d) and pBN56 (FIG.3 e), comprising about 600 bps of MVA sequences flanking the insertionsite between the ORF 007R and 008L (Flank 1: F1 IGR 07-08 starts atposition 12200 of the MVA genome; Flank 2: F2 IGR 07-08 stops atposition 13400 of the MVA genome). F2rpt stands for a repetitivesequence of Flank 2 to allow deletion of the reporter cassette from aresulting recombinant virus. Additionally the vector pBNX88 (FIG. 3 c)and pBNX86 (FIG. 3 b) comprise exogenous DNA (BFP+gpt and NPT II+EGFP,respectively) under the transcriptional control of a poxvirus promoterP) between the flanking sequences. F2rpt stands for a repetitivesequence of Flank 2 to allow deletion of the reporter cassette from aresulting recombinant virus. PBN56 (FIG. 3 e) additionally codes for theHIV-1 env protein, and pBN34 (FIG. 3 d) contains the Denguevirus PrM2coding sequence under control of a poxvirus promoter. Furtherabbreviations: AmpR=Ampicilin resistance gene; bps=base pairs.

FIG. 4: Restriction map of the vector constructs pBNX80 (FIG. 4 a),pBNX87 (FIG. 4 b) and pBN47 (FIG. 4 c) comprising about 600/640 bps ofMVA sequences flanking the insertion site between the ORF 044L and 045L(Flank 1: F1 IGR44-45 starts at position 36730 of the MVA genome; Flank2: F2 IGR44-45 stops at position 37970 of the MVA genome). Additionallythe vector pBNX87 (FIG. 4 b) comprises exogenous DNA (NPT II gene+EGFP)under the transcriptional control of a poxvirus promoter P) between theflanking sequences. F2rpt stands for a repetitive sequence of Flank 2 toallow deletion of the reporter cassette from a resulting recombinantvirus. PBN47 (FIG. 4 c) additionally codes for the Denguevirus PrM3under the control of a poxvirus promoter.

Further abbreviations: AmpR=Ampicilin resistance gene; bps=base pairs.

FIG. 5: Restriction map of the vector constructs pBNX90 (FIG. 5 a),pBNX92 (FIG. 5 b) and pBN54 (FIG. 5 c), comprising about 596/604 bps ofMVA sequences flanking the insertion site between the ORF 148R and 149L(Flank 1: F1 IGR148-149 starts at position 136900 of the MVA genome;Flank 2: F2 IGR148-149 stops at position 138100 of the MVA genome).Additionally the vector pBNX92 (FIG. 5 b) comprises exogenous DNA(gpt+BFP) under the transcriptional control of a poxvirus promoter P)between the flanking sequences. PBN54 (FIG. 5 c) additionally codes forthe Denguevirus PrM1. F2rpt stands for a repetitive sequence of Flank 2to allow deletion of the reporter cassette from a resulting recombinantvirus. Further abbreviations: AmpR=Ampicilin resistance gene; bps=basepairs.

FIG. 6: Schematic presentation of the intergenic insertion sites of MVA(Genbank Ac. U94848).

FIG. 7: PCR analysis of IGR I4L-I5L in recombinant MVA with theDenguevirus NS1 inserted in the IGR I4L-I5L. Lane “BN” shows the PCRproduct using MVA-BN empty vector. Using the NS1 recombinant MVA afragment of bigger size is detectable (1, 2, 3, 4: differentconcentrations of DNA). M=Molecular weight marker, H₂O=water negativecontrol.

FIG. 8: FIG. 8 a: PCR analysis of IGR 136-137 in recombinant MVA withthe Denguevirus PrM4 inserted in the IGR 136-137. Lane “BN” shows thePCR product using MVA-BN empty vector. Using the PrM4 recombinant MVA afragment of bigger size is detectable (mBN23, 1/10, 1/100: differentconcentrations of DNA). M=Molecular weight marker, H₂O=water negativecontrol, pBN27=plasmid positive control.

FIG. 8 b: multiple step growth curve for MVA-BN empty vector and therecombinant MVA with PrM4 inserted in IGR 136-137 (MVA-mBN23).

FIG. 9: FIG. 9 a: PCR analysis of IGR 07-08 in recombinant MVA with theDenguevirus PrM2 inserted in the IGR 07-08. Lane 3 shows the PCR productusing MVA-BN empty vector. Using the PrM2 recombinant MVA a fragment ofbigger size is detectable (lane 2). M=Molecular weight marker, lane1=water negative control.

FIG. 9 b: multiple step growth curve for MVA-BN empty vector and therecombinant MVA with PrM2 inserted in IGR 07-08 (MVA-mBN25).

FIG. 10: PCR analysis of IGR 07-08 in recombinant MVA with the HIV envinserted in the IGR 07-08. Lane BN shows the PCR product using MVA-BNempty vector. Using the PrM2 recombinant MVA a fragment of bigger sizeis detectable (lane 1, 2, 3). M=Molecular weight marker, −=waternegative control, +=plasmid positive control.

FIG. 11: FIG. 11 a: PCR analysis of IGR 44-45 in recombinant MVA withthe Denguevirus PrM3 inserted in the IGR 44-45. Lane BN shows the PCRproduct using MVA-BN empty vector. Using the PrM3 recombinant MVA afragment of bigger size is detectable (lane 1-4 different concentrationsof DNA). M=Molecular weight marker, −=water negative control.

FIG. 11 b: multiple step growth curve for MVA-BN empty vector and therecombinant MVA with PrM3 inserted in IGR 44-45 (MVA-mBN28).

FIG. 12: FIG. 12 a: PCR analysis of IGR 148-149 in recombinant MVA withthe Denguevirus PrM1 inserted in the IGR 148-149. Lane BN shows the PCRproduct using MVA-BN empty vector. Using the PrM1 recombinant MVA afragment of bigger size is detectable (lane 1). M=Molecular weightmarker, −=water negative control, +=plasmid positive control.

FIG. 12 b: multiple step growth curve for MVA-BN empty vector and therecombinant MVA with PrM1 inserted in IGR 44-45 (MVA-mBN33).

The following examples will further illustrate the present invention. Itwill be well understood by any person skilled in the art that theprovided examples in no way are to be interpreted in a way that limitsthe present invention to these examples. The scope of the invention isonly to be limited by the full scope of the appended claims.

Example 1 Insertion Vectors pBNX39, pBNX70 and pBN84

For the insertion of exogenous sequences into the intergenic regionadjacent to the 065L ORF (insertion site is at genome position 56760) ofMVA, a vector was constructed which comprises about 1200 bp of theflanking sequences adjacent to the insertion site. These flankingsequence are separated into two flanks comprising on one flank about 610bp of the 065L ORF (alternative nomenclature: I4L ORF) and on the otherpart about 580 bp of the intergenic region behind the 065L ORF as wellas parts of the proximate ORF. In between these flanking sequences anEcogpt gene (gpt stands for phosphoribosyltransferase gene isolated fromE. coli) and a BFP (blue fluorescence protein), respectively, is locatedunder the transcriptional control of a poxyiral promoter. Additionally,there is at least one cloning site for the insertion of additional genesor sequences to be inserted into the intergenic region behind the I4LORF. Exemplary vector constructs according to the present invention aredisclosed in FIG. 1 a) and b) (pBNX39, pBNX70). In vector pBN84 (FIG. 1c) the coding region for Denguevirus NS1 is inserted in the cloning siteof pBNX70 (FIG. 1 b).

Generation of the Recombinant MVA Via Homologous Recombination

Foreign genes can be inserted into the MVA genome by homologousrecombination. For that purpose the foreign gene of interest is clonedinto a plasmid vector, as described above. This vector is transfected inMVA infected cells. The recombination takes place in the cytoplasm ofthe infected and transfected cells. With help of the selection and/orreporter cassette, which is also contained in the insertion vector,cells comprising recombinant viruses are identified and isolated.

For homologous recombination BHK (Baby hamster kidney) cells or CEF(primary chicken embryo fibroblasts) are seeded in 6 well plates usingDMEM (Dulbecco's Modified Eagles Medium, Gibco BRL)+10% fetal calf serum(FCS) or VP-SFM (Gibco BRL)+4 mmol/l L-Glutamine for a serum freeproduction process.

Cells need to be still in the growing phase and therefore should reach60-80% confluence on the day of transfection. Cells were counted beforeseeding, as the number of cells has to be known for determination of themultiplicity of infection (moi) for infection.

For the infection the MVA stock is diluted in DMEM/FCS orVP-SFM/L-Glutamine so that 500 μl dilution contain an appropriate amountof virus that will give a moi of 0.1-1.0. Cells are assumed to havedivided once after seeding. The medium is removed from cells and cellsare infected with 500 μl of diluted virus for 1 hour rocking at roomtemperature. The inoculum is removed and cells are washed withDMEM/VP-SFM. Infected cells are left in 1.6 ml DMEM/FCS andVP-SFM/L-Glutamine, respectively, while setting up the transfectionreaction (Qiagen Effectene Kit).

For the transfection, the “Effectene” transfection kit (Qiagen) is used.A transfection mix is prepared of 1-2 μg of linearized insertion vector(total amount for multiple transfection) with buffer EC to give a finalvolume of 100 μl. Add 3.2 μl Enhancer, vortex and incubate at roomtemperature for 5 min. Then, 10 μl of Effectene are added aftervortexing stock tube and the solution is mixed thoroughly by vortexingand incubated at room temperature for 10 min. 600 μl of DMEM/FCS andVP-SFM/L-Glutamine respectively, are added, mixed and subsequently, thewhole transfection mix is added to the cells, which are already coveredwith medium. Gently the dish is rocked to mix the transfection reaction.Incubation takes place at 37° C. with 5% CO₂ over night. The next daythe medium is removed and replaced with fresh DMEM/FCS orVP-SFM/L-Glutamine. Incubation is continued until day 3.

For harvesting, the cells are scraped into medium, then the cellsuspension is transferred to an adequate tube and frozen at −20° C. forshort-term storage or at −80° C. for long-term storage.

Insertion of Ecogpt in the I4L Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol and were additionally transfected withinsertion vector pBNX39 (FIG. 1 a) containing the Ecogpt gene (Ecogpt,or shortened to gpt, stands for phosphoribosyltransferase gene) asreporter gene. Resulting recombinant viruses were purified by 3 roundsof plaque purification under phosphribosyl-transferase metabolismselection by addition of mycophenolic acid, xanthin and hypoxanthin.Mycophenolic acid (MPA) inhibits inosine monophosphate dehydrogenase andresults in blockage of purine synthesis and inhibition of viralreplication in most cell lines. This blockage can be overcome byexpressing Ecogpt from a constitutive promoter and providing thesubstrates xanthine and hypoxanthine.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the I4L insertion side primer pair, BN499 (CAA CTC TCT TCTTGA TTA CC, SEQ ID NO.: 1) and BN500 (CGA TCA AAG TCA ATC TAT G, SEQ IDNO.: 2) were used. In case the DNA of the empty vector virus MVA isamplified the expected PCR fragment is 328 nucleotides (nt) long, incase a recombinant MVA is amplified, which has incorporated exogenousDNA at the I4L insertion site, the fragment is correspondingly enlarged.

Insertion of NS1 in the IGR064L-065L (I4L-I5L) Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol and were additionally transfected withinsertion vector pBN84 (FIG. 1 c) containing the Ecogpt gene forselection and BFP (Blue fluorescence protein) as reporter gene.Resulting recombinant viruses were purified by 7 rounds of plaquepurification under phosphribosyl-transferase metabolism selection byaddition of mycophenolc acid, xanthin and hypoxanthin. Mycophenolic acid(MPA) inhibits inosine monophosphate dehydrogenase and results inblockage of purine synthesis and inhibition of viral replication in mostcell lines. This blockage can be overcome by expressing Ecogpt from aconstitutive promoter and providing the substrates xanthine andhypoxanthine.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the I4L insertion side primer pair, BN499 (CAA CTC TCT TCTTGA TTA CC, SEQ ID NO.: 1) and BN500 (CGA TCA AAG TCA ATC TAT G, SEQ IDNO.: 2) were used. In case the DNA of the empty vector virus MVA isamplified the expected PCR fragment is 328 nucleotides (nt) long, incase a recombinant MVA for NS1 is amplified, which has incorporatedDenguevirus NS1 coding region at the I4L insertion site, the fragment isexpected to be 1683 bp. The PCR results in FIG. 7 show clearly thestable insertion of NS1 in the I4L insertion site after 17 rounds ofvirus amplification.

Testing of recMVA Including NS1 (MVA-BN22) In Vitro

A T25 flask with about 80% confluent monolayers of BHK cells wasinoculated with 100 μl of the virus stock diluted to 1×10⁷ in MEMα with1% FCS and rocked at room temperature for 30 minutes. 5 ml of MEMα with3% FCS was added to each flask and incubated at 30° C. in a CO₂incubator. The flask was harvested after 48 hours. The supernatant wasremoved from the flask and spun at 260 g for 10 minutes at 4° C. Thesupernatants were stored in aliquots at −80° C. The pellet was washedwith 5 ml of 1×PBS twice and then resuspended in 1 ml of hypotonicdouncing buffer with 1% TX100. The cell lysates were harvested and spunfor 5 minutes at 16,000 g and the supernatants were stored in amicrocentrifuges tube at −80° C.

Flasks inoculated with MVA including GFP, MVA including the NS1 gene ina deletion site (MVA-BN07), and mock infected flasks were also treatedthe same way as described above.

The cell/viral lysate and the supernatant were treated innon-reducing/reducing sample buffer under non-heated/heated conditions.The proteins were separated on 10% SDS PAGE and transferred tonitrocellulose membranes. The blots were probed overnight with pooledconvalescent patients' sera (PPCS) at 1:500 dilution. After washing 3times with 1×PBS the blots were incubated with anti-human IgG-HRP (DAKO)for 2 hours at room temperature. After the blots were washed asdescribed before, the colour was developed using 4 chloro-1-naphtol.

The western blot results showed that NS1 in MVA-BN22 is expressed inlarge quantities. NS1 was expressed in the right confirmation, as adimer under non-heated condition and as a monomer under heatedcondition.

The NS1 expression was compared in both MVA-BN22 and MVA-BN07. The BHKcells were inoculated with the same pfu and harvested after 48 hours.The results showed that the expression of NS1 was much higher in BN22than in BN07. The western blots results also showed that there is moreNS1 secreted in the supernatant with the BN22 construct compared toBN07.

The results also showed that NS1 expressed in cells infected with BN22is antigenic and is recognized by the pooled convalescent patients'sera.

In conclusion, NS1 is expressed in large quantities and in the rightconfirmation in the BHK cells infected with BN22. Both the dimer andmonomer are antigenic and are recognized by the pooled convalescentpatients' sera.

Example 2 Insertion Vector pBNX67 and pBN27

The MVA sequences adjacent the new insertion site (at genome position129940) between the ORF 136L and 137L were isolated by standard PCRamplification of the sequence of interest using the following primers:

oBN543 (TCCCCGCGGAGAGGCGTAAAAGTTAAATTAGAT; SEQ ID NO.: 3) and oBN544(TGATCTAGAATCGCTCGTAAAAACTGCGGAGGT; SEQ ID NO.: 4)for isolating Flank 1; oBN578(CCGCTCGAGTTCACGTTCAGCCTTCATGC; SEQ ID NO.: 5) and oBN579(CGGGGGCCCTATTTTGTATAATATCTGGTAAG; SEQ ID NO.: 6) for isolating Flank 2.

The PCR fragment comprising Flank 1 was treated with the restrictionenzymes SacII and XbaI and ligated to, a SacII/XbaI digested anddephosphorylated basic vector, such as pBluescript (Stratagene).

The resulting plasmid was XhoI/ApaI digested, dephosphorylated andligated to the XhoI/ApaI digested PCR fragment comprising Flank 2.

Optionally, a repetitive sequence of Flank 2 which had been isolated byPCR using the primers oBN545 (CGGCTGCAGGGTACCTTCACGTTCAGCCTTCATGC; SEQID NO.: 7) and oBN546 (CGGAAGCTTTATATGGTTTAGGATATTCTGTTTT; SEQ ID NO.:8) and which became HindIII/PstI digested, was inserted into theHindIII/PstI site of the resulting vector. FIG. 2 a) shows the vector(pBNX51).

A reporter cassette comprising a synthetic promoter, NPT II gene(neomycin resistance), poly-A region, IRES, EGFP gene(Ps-NPTII-polyA-IRES-EGFP) was Ec1136II/XhoI digested and inserted intothe HindIII/XhoI site of the insertion vector, wherein the HindIII sitewas blunt ended with T4 DNA Polymerase (Roche). A restriction map of anexemplary vector construct according to this example is disclosed inFIG. 2 b) (pBNX67).

For construction of pBN27 (FIG. 2 c)the Denguevirus PrM of serotype 4was inserted in the single Pad site of pBNX67.

Generation of the Recombinant MVA Via Homologous Recombination

The vector pBNX67 (FIG. 2 b) can be used to generate a recombinant MVAusing the above mentioned protocol—e.g. using pBN27 (FIG. 2 c) forhomologous recombination results in a recombinant MVA carryingDenguevirus PrM4 in the intergenic region between two adjacent ORFs.

Insertion of PrM4 in the IGR136-137 Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol and were additionally transfected withinsertion vector pBN27 (FIG. 2 c) containing the NPT gene for selectionand EGFP (enhanced green fluorescence protein) as reporter gene.Resulting recombinant viruses were purified by 4 rounds of plaquepurification under G418 selection.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the IGR136-137 insertion side primer pair, BN900(cgttcgcatgggttacctcc, SEQ ID NO.: 9) and BN901 (gacgcatgaaggctgaac, SEQID NO.: 10) were used. In case the DNA of the empty vector virus MVA isamplified the expected PCR fragment is 88 nucleotides (nt) long, in casea recombinant MVA for PrM4 is amplified, which has incorporatedDenguevirus PrM4 coding region at the IGR136-137 insertion site, thefragment is expected to be 880 bp. The PCR results in FIG. 8 a) showclearly the stable insertion of PrM4 in the IGR136-137 insertion siteafter 22 rounds of virus amplification. The recombinant MVA still showsthe same growth characteristics as MVA-BN. It replicates in chickenembryo fibroblasts (CEF cells) and grows attenuated in mammalian cells(FIG. 8 b).

Example 3 Insertion Vector pBNX79, pBNX86, pBNX88, pBN34 and pBN56

The MVA sequences adjacent the new insertion site (at genome position12800) between the ORF 007R and 008L were isolated by standard PCRamplification of the sequence of interest using the following primers:

IGR 07/08 F1up (CGCGAGCTCAATAAAAAAAAGTTTTAC; SEQ ID NO.: 11) andIGR 07/08 F1end (AGGCCGCGGATGCATGTTATGCAAAATAT; SEQ ID NO.: 12)for isolating Flank 1; IGR 07/08 F2up(CCGCTCGAGCGCGGATCCCAATATATGGCATAGAAC;  SEQ ID NO.: 13) andIGR 07/08 F2end (CAGGGCCCTCTCATCGCTTTCATG; SEQ ID NO.: 14)for isolating Flank 2.

The PCR fragment comprising Flank 1 was treated with the restrictionenzymes SacII and SacI and ligated to a SacII/SacI digested anddephosphorylated basic vector, such as pBluescript (Stratagene).

The resulting plasmid was XhoI/ApaI digested, dephosphorylated andligated to the XhoI/ApaI digested PCR fragment comprising Flank 2.

Optionally, a repetitive sequence of Flank 2 which had been isolated byPCR using the primers IGR 07/08 F2up(CCGCTCGAGCGCGGATCCCAATATATGGCATAGAAC; SEQ ID NO.: 13) and IGR 07/08F2mid (TTTCTGCAGTGATATTTATCCAATACTA; SEQ ID NO.: 15) and which isBamHI/PstI digested, was inserted into the BamHI/PstI site of theresulting vector.

Any reporter or therapeutical gene comprising cassette, having e.g. apoxyiral promoter, a marker gene, a poly-A region and optionally an IRESelement, a further gene, e.g. expressing a therapeutically activesubstance or gene product, can be blunt ended with T4 DNA Polymerase(Roche) after a restriction digest and inserted into a suitable cloningsite of the plasmid vector. A restriction map of an exemplary vectorconstruct according to this example is disclosed in FIG. 3 a) (pBNX79).Insertion of the NPT/EGFP selection cassette resulted in vector pBNX86(FIG. 3 b) and insertion of the gpt/BFP selection cassette in vectorpBNX88 (FIG. 3 c), respectively. Considering an expression unit for atherapeutic gene, comprising a therapeutic gene and an operably linkedpromoter, this expression unit is inserted into the Pad site. Forconstruction of pBN34 (FIG. 3 d) the Denguevirus PrM2 was cloned inpBNX88 (FIG. 3 c) and for synthesis of pBN56 (FIG. 3 e) the HIV envcoding region was Pad cloned in pBNX86 (FIG. 3 b).

Generation of the Recombinant MVA Via Homologous Recombination

The vectors pBNX86 (FIG. 3 b) and pBNX88 (FIG. 3 c), respectively, canbe used to generate a recombinant MVA using the above mentionedprotocol. Using pBN34 (FIG. 3 d) for homologous recombination results ina recombinant MVA carrying Denguevirus PrM2 in the intergenic regionbetween two adjacent ORFs. Recombination of pBN56 (FIG. 3 e) with theMVA-BN genome results in a recombinant MVA, which contains the HIV envgene in the corresponding IGR.

Insertion of PrM2 in the IGR07-08 Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol and were additionally transfected withinsertion vector pBN34 (FIG. 3 d) containing the gpt gene for selectionand BFP as reporter gene. Resulting recombinant viruses were purified by3 rounds of plaque purification under selection by mycophenolic acid asdescribed in Example 1.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the IGR07-08 insertion side primer pair, BN902(ctggataaatacgaggacgtg, SEQ ID NO.: 16) and BN903 (gacaattatccgacgcaccg,SEQ ID NO.: 17) were used. In case the DNA of the empty vector virus MVAis amplified the expected PCR fragment is 190 nucleotides (nt) long, incase a recombinant MVA for PrM2 is amplified, which has incorporatedDenguevirus PrM2, coding region at the IGR07-08 insertion site, thefragment is expected to be 950 bp. The PCR results in FIG. 9 a) showclearly the stable insertion of PrM2 in the IGR07-08 insertion siteafter 20 rounds of virus amplification. The recombinant MVA still showsthe same growth characteristics as MVA-BN. It replicates in chickenembryo fibroblasts (CEF cells) and grows attenuated in mammalian cells(FIG. 9 b).

Insertion of HIV Env in the IGR07-08 Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol and were additionally transfected withinsertion vector pBN56 (FIG. 3 e) containing the NPT gene for selectionand EGFP as reporter gene. Resulting recombinant viruses were purifiedby 6 rounds of plaque purification under G418 selection.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the IR07-08 insertion side primer pair, BN902(ctggataaatacgaggacgtg, SEQ ID NO.: 16) and BN903 (gacaattatccgacgcaccg,SEQ ID NO.: 17) were used. In case the DNA of the empty vector virus MVAis amplified the expected PCR fragment is 190 nucleotides (nt) long, incase a recombinant MVA for env is amplified, which has incorporated HIVenv coding region at the IGR07-08 insertion site, the fragment isexpected to be 2.6 kb. The PCR results in FIG. 10 show clearly thestable insertion of env in the IGR07-08 insertion site after 20 roundsof virus amplification.

Example 4 Insertion Vector pBNX80, pBNX87 and pBN47

The MVA sequences adjacent the new insertion site (at genome position37330) between the ORF 044L and 045L were isolated by standard PCRamplification of the sequence of interest using the following primers:

IGR44/45F1up (CGCGAGCTCATTTCTTAGCTAGAGTGATA; SEQ ID NO.: 18) andIGR44/45F1end (AGGCCGCGGAGTGAAAGCTAGAGAGGG; SEQ ID NO.: 19)for isolating Flank 1; IGR44/45F2up(CCGCTCGAGCGCGGATCCTAAACTGTATCGATTATT; SEQ ID NO.: 20) and IGR44/45F2end(CAGGGCCCCTAAATGCGCTTCTCAAT; SEQ ID NO.: 21) for isolating Flank 2.

The PCR fragment comprising Flank 1 was treated with the restrictionenzymes SacII and SacI and ligated to a SacII/SacI digested anddephosphorylated basic vector, such as pBluescript (Stratagene).

The resulting plasmid was XhoI/ApaI digested, dephosphorylated andligated to the XhoI/ApaI digested PCR fragment comprising Flank 2.

Optionally, a repetitive sequence of Flank 2, which had been isolated byPCR using the primers IGR44/45F2up(CCGCTCGAGCGCGGATCCTAAACTGTATCGATTATT; SEQ ID NO.: 20) and IGR44/45F2mid(TTTCTGCAGCCTTCCTGGGTTTGTATTAACG; SEQ ID NO.: 22) and which becameBamHI/PstI digested, was inserted into the BamHI/PstI site of theresulting vector.

Any reporter or therapeutical gene comprising cassette, having e.g. apoxyiral promoter, a marker gene, a poly-A region and optionally an IRESelement, a further gene, e.g. expressing a therapeutically activesubstance or gene product, can be blunt ended with T4 DNA Polymerase(Roche) after an restriction digest and inserted into a suitable cloningsite of the plasmid vector. Considering a reporter gene cassette thePstI, EcoRI, EcoRV, HindIII, AvaI or XhoI restriction enzyme sitebetween Flank 2 and the Flank-2-repetition is preferred as cloning site.For the construction of pBNX87 (FIG. 4 b) the NPT/EGFP selectioncassette was inserted in pBNX80 (FIG. 4 a). Considering an expressionunit for a therapeutic gene, comprising a therapeutic gene and anoperably linked promoter, this expression unite is inserted into the Padsite.

A restriction map of exemplary vector constructs according to thisexample are disclosed in FIG. 4 a) and b) (pBNX80, pBNX87).

The vector can be used to generate a recombinant MVA—following theabove-mentioned protocol—carrying an exogenous sequence in theintergenic region between two adjacent ORFs. For the construction ofpBN47 (FIG. 4 c) the PrM of Denguevirus serotype 3 was cloned intopBNX87 (FIG. 4 b).

Insertion of PrM3 in the IGR44-45 Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol and were additionally transfected withinsertion vector pBN47 (FIG. 4 c) containing the NPT gene for selectionand EGFP as reporter gene. Resulting recombinant viruses were purifiedby 3 rounds of plaque purification under G418 selection.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the IGR44-45 insertion side primer pair, BN904(cgttagacaacacaccgacgatgg, SEQ ID NO.: 23) and BN905(cggatgaaaaatttttggaag, SEQ ID NO.: 24) were used. In case the DNA ofthe empty vector virus MVA is amplified the expected PCR fragment is 185nucleotides (nt) long, in case a recombinant MVA for PrM3 is amplified,which has incorporated Denguevirus PrM3 coding region at the IGR44-45insertion site, the fragment is expected to be 850 bp. The PCR resultsin FIG. 11 a) show clearly the stable insertion of PrM3 in the IGR44-45insertion site after 19 rounds of virus amplification. The recombinantMVA still shows the same growth characteristics as MVA-BN. It replicatesin chicken embryo fibroblasts (CEF cells) and grows attenuated inmammalian cells (FIG. 11 b).

Example 5 Insertion Vector pBNX90, pBNX92 and pBN54

The MVA sequences adjacent the new insertion site (at genome position137496) between the ORF 148R and 149L were isolated by standard PCRamplification of the sequence of interest using the following primers:

IGR148/149F1up (TCCCCGCGGGGACTCATAGATTATCGACG; SEQ ID NO.: 25) andIGR148/149F1end (CTAGTCTAGACTAGTCTATTAATCCACAGAAATAC; SEQ ID NO.: 26)for isolating Flank 1; IGR148/149F2up(CCCAAGCTTGGGCGGGATCCCGTTTCTAGTATGGGGATC; SEQ ID NO.: 27) andIGR148/149F2end (TAGGGCCCGTTATTGCCATGATAGAG; SEQ ID NO.: 28)for isolating Flank 2.

The PCR fragment comprising Flank 1 was treated with the restrictionenzymes SacII and XbaI and ligated to a SacII/XbaI digested anddephosphorylated basic vector, such as pBluescript (Stratagene).

The resulting plasmid was HindIII/ApaI digested, dephosphorylated andligated to the HindIII/ApaI digested PCR fragment comprising Flank 2.

Optionally, a repetitive sequence of Flank 2, which had been isolated byPCR using the primers IGR148/149F2up(CCCAAGCTTGGGCGGGATCCCGTTTCTAGTATGGGGATC; SEQ ID NO.: 27) andIGR148/149F2mid (TTTCTGCAGTGTATAATACCACGAGC; SEQ ID NO.: 29) and whichbecame BamHI/PstI digested, was inserted into the BamHI/PstI site of theresulting vector.

Any reporter or therapeutical gene comprising cassette, having e.g. apoxyiral promoter, a marker gene, a poly-A region and optionally an IRESelement, a further gene, e.g. expressing a therapeutically activesubstance or gene product, can be blunt ended with T4 DNA Polymerase(Roche) after an restriction digest and inserted into a suitable cloningsite of the plasmid vector. For construction of pBNX92 (FIG. 5 b) thegpt/BFP expression cassette was inserted in this cloning site.Considering a reporter gene cassette the PstI, EcoRI, EcoRV and HindIIIrestriction enzyme site between Flank 2 and the Flank-2-repetition ispreferred as cloning site. Considering an expression unit for atherapeutic gene, comprising a therapeutic gene and an operably linkedpromoter, this expression unite is inserted into the PacI site. Forconstruction of pBN54 (FIG. 5 c) the Denguevirus PrM1 was inserted inthis Pad site.

A restriction map of an exemplary vector construct according to thisExample is disclosed in FIG. 5 a) and b) (pBNX90, pBNX92).

The vector can be used to generate a recombinant MVA—following theabove-mentioned protocol—carrying an exogenous sequence in theintergenic region between two adjacent ORFs. For the generation of arecombinant MVA expressing the Denguevirus PrM1 pEN54 (FIG. 5 c) wasused for a homologous recombination.

Insertion of PrM1 in the IGR148-149 Insertion Site of MVA

In a first round, cells were infected with MVA according to theabove-described protocol and were additionally transfected withinsertion vector pEN54 (FIG. 5 c) containing the gpt gene for selectionand BFP as reporter gene. Resulting recombinant viruses were purified by3 rounds of plaque purification under selection with mycophenolic acid.

Resulting recombinant viruses were identified by standard PCR assaysusing a primer pair selectively amplifying the expected insertion site.To amplify the IGR148-149 insertion side primer pair, BN960(ctgtataggtatgtcctctgcc, SEQ ID NO.: 30) and BN961 (gctagtagacgtggaaga,SEQ ID NO.: 31) were used. In case the DNA of the empty vector virus MVAis amplified the expected PCR fragment is 450 nucleotides (nt) long, incase a recombinant MVA for PrM1 is amplified, which has incorporatedDenguevirus PrM1 coding region at the IGR148-149 insertion site, thefragment is expected to be 1200 bp. The PCR results in FIG. 12 a) showclearly the stable insertion of PrM1 in the IGR148-149 insertion siteafter 23 rounds of virus amplification. The recombinant MVA still showsthe same growth characteristics as MVA-BN. It replicates in chickenembryo fibroblasts (CEF cells) and grows attenuated in mammalian cells(FIG. 12 b).

1. A method for producing a recombinant modified vaccinia Ankara viruscomprising: (a) transfecting a cell with a plasmid vector comprising anintergenic region (IGR) of the viral genome, wherein inserted into saidIGR is a heterologous DNA sequence, wherein the IGR is not a naturallyoccurring deletion site or the tk-locus; (b) infecting the transfectedcell with an MVA; and (c) isolating a recombinant MVA comprising theheterologous DNA.
 2. The method of claim 1, wherein the IGR is IGR007R-008L.
 3. The method of claim 1, wherein the IGR is IGR 018L-019L.4. The method of claim 1, wherein the IGR is IGR 044L-045L.
 5. Themethod of claim 1, wherein the IGR is IGR 064L-065L.
 6. The method ofclaim 1, wherein the IGR is IGR 148R-149L.
 7. The method of claim 1,wherein the heterologous DNA sequence is placed under thetranscriptional control of a poxviral transcription control element. 8.The method of claim 7, wherein the poxviral transcription controlelement is an ATI promoter.
 9. The method of claim 1, wherein theheterologous DNA sequence encodes at least one cancer antigen.
 10. Themethod of claim 9, wherein the cancer antigen is inserted into an IGRselected from the group consisting of IGRs 007R-008L, 018L-019L,044L-045L, 064L-065L, and 148R-149L.
 11. The method of claim 10, whereinthe cancer antigen is inserted into IGR 064L-065L.
 12. The method ofclaim 1, wherein the heterologous DNA sequence comprises a sequence froman infectious virus.
 13. The method of claim 12, wherein theheterologous DNA sequence is inserted into IGR 007R-008L.
 14. The methodof claim 12, wherein the heterologous DNA sequence is inserted into IGR018L-019L.
 15. The method of claim 12, wherein the heterologous DNAsequence is inserted into IGR 044L-045L.
 16. The method of claim 12,wherein the heterologous DNA sequence is inserted into IGR 064L-065L.17. The method of claim 12, wherein the heterologous DNA sequence isinserted into IGR 148R-149L.
 18. The method of claim 12, wherein theheterologous DNA sequence comprises a sequence from Dengue virus,Japanese encephalitis virus, Hepatitis virus B, Hepatitis virus C, orhuman immunodeficiency virus (HIV).
 19. The method of claim 18, whereinthe heterologous DNA sequence comprises a sequence from Dengue virus.20. The method of claim 19, wherein the Dengue virus sequence isselected from the group consisting of NS1 and PrM sequences.
 21. Themethod of claim 20, wherein the PrM sequence is inserted into an IGRselected from the group consisting of IGRs 007R-008L, 018L-019L,044L-045L, 064L-065L, and 148R-149L.
 22. The method of claim 20, whereinthe NS1 sequence is inserted into an IGR selected from the groupconsisting of IGRs 007R-008L, 018L-019L, 044L-045L, 064L-065L, and148R-149L.